Rickettsia conorii is a potent complement activator in vivo and combined inhibition of complement and CD14 is required for attenuation of the cytokine response ex vivo.

Mediterranean spotted fever caused by Rickettsia conorii is a potentially lethal disease characterized by vascular inflammation affecting multiple organs. Studies of R. conorii so far have focused on activation of inflammatory cells and their release of inflammatory cytokines, but complement activation has not been investigated in R. conorii-infected patients. Here, we performed a comprehensive analysis of complement activation markers and the soluble cross-talking co-receptor CD14 (sCD14) in plasma from R. conorii-infected patients. The clinical data were supplemented with ex vivo experiments where the cytokine response was characterized in human whole blood stimulated with R. conorii. Complement activation markers at the level of C3 (C3bc, C3bBbP) and terminal pathway activation (sC5b-9), as well as sCD14, were markedly elevated (p <0.01 for all), and closely correlated (p <0.05 for all), in patients at admission compared with healthy matched controls. All tested markers were significantly reduced to baseline values at time of follow up. Rickettsia conorii incubated in human whole blood was shown to trigger complement activation accompanied by release of the inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, IL-8 and tumour necrosis factor. Whereas inhibition of either C3 or CD14 had only a minor effect on released cytokines, combined inhibition of C3 and CD14 resulted in significant reduction, virtually to baseline levels, of the four cytokines (p <0.05 for all). Our data show that complement is markedly activated upon R. conorii infection and complement activation is, together with CD14, responsible for a major part of the cytokine response induced by R. conorii in human whole blood.

Effects of single applications of 12-O-tetradecanoylphorbol-13-acetate, mezerein, or ethylphenylpropiolate on DNA synthesis and polyamine levels in hairless mouse epidermis.

Quantitative and qualitative changes in epidermal polyamine levels and DNA synthesis (specific activity and labeling index) after a single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein (MEZ), or ethylphenylpropiolate (EPP) in acetone were studied concomitantly in the same epidermal cell population from each treated mouse. The doses tested were 17 nmol of TPA; 1.7, 8.5, and 17 nmol of MEZ; and 20 mumol of EPP. With relatively small variations in time patterns, both the tumor promoter TPA and the mitogens MEZ and EPP caused similar sequential changes: initial inhibition of DNA synthesis; induction of L-ornithine decarboxylase activity; and subsequent peaks of putrescine levels preceding peaks in the rate of DNA synthesis. A remarkably good correlation between the molar ratio of spermidine/spermine and the increase in DNA synthesis was seen after all three of the compounds. However, in the initial period with inhibited DNA synthesis, a negative correlation between spermidine/spermine and DNA synthesis was observed after all of the treatments. TPA and MEZ induced pronounced biphasic increases in DNA synthesis, accumulation of putrescine, and the spermidine/spermine ratio, whereas EPP induced single-peaked increases in the same variables. The fluctuations in polyamine levels and DNA synthesis were associated with cohorts of partly synchronized cells passing the cell cycle multiple turns. Thus, the induction of L-ornithine decarboxylase and the polyamines does not seem to be specific for tumor promotion but merely seems to be associated with the cell kinetic events during stimulated cell proliferation. It is suggested that quantitative aspects of hyperproliferation may be essential for tumor promotion.

The tumourigenic and carcinogenic effect of 12-0-tetradecanoylphorbol-13-acetate when applied to the skin of BALB/cA and hairless (hr/hr) mice.

One hundred and fourteen BALB/cA and 89 hairless (hr/hr, Oslo strain) mice of both sexes were treated topically once a week on the dorsal skin for about 17 months with 18 nmol 12-0-tetradecanoylphorbol-13-acetate (TPA) in 0.2 ml acetone or with 0.2 ml acetone alone, and observed for up to 24 months. At autopsy all visible lesions and selected areas from the skin, nasal cavities and a number of internal organs were examined microscopically. Only TPA-treated animals developed epidermal tumours, i.e. squamous cell papillomas and carcinomas, which were the only tumours with a higher incidence in TPA-treated animals than in controls. The BALB/cA mice developed more epidermal tumours than the hr/hr mice. Other tumours were: reticuloses, malignant lymphomas, lung adenomas, subcutaneous fibrosarcomas, parenchymal adenomas, hemangiomas and one angiosarcoma of the liver, intestinal adenocarcinomas and one granulosa cell tumour of the ovary. Various degrees of amyloidosis in several organs, and subcapsular fusiform cell hyperplasia in the adrenal glands, occurred in both mouse strains. Kupffer cell proliferation was more prevalent in the hr/hr mice treated with TPA than in the controls. This lesion was absent in the BALB/cA strain. Hence, TPA is a weak skin carcinogen for both BALB/cA and hr/hr mice, and multiple skin applications of the dose of TPA used do not alter the frequency of spontaneous internal tumours in these mouse strains.

Cell population kinetics in hairless mouse epidermis following a single topical application of 12-O-tetradecanoylphorbol-13-acetate II.

It is known that a single application of 17 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) to hairless mouse skin first induces (from 0-12 h) a short block in mitotic activity followed by a transient stimulation of proliferation (from 12-96 h) characterized by multiple waves of rapid proliferation in partly synchronized basal cells, leading to hyperplasia. The replication rate of basal cells, the number of basal and suprabasal (maturing) cells per unit of interfollicular epidermis and the number of squamous layers in the stratum corneum were recorded. Changes in the nuclear area of living cells were monitored by morphometry. The changes in the rate of basal cell proliferation are accompanied by concomitant waves of increased rates of cell maturation and cell loss, with a considerable reduction in epidermal cell transit time. When 14-15 squamous layers were observed, this resulted in an increased cell loss, which was visible as scaling from 24 h onwards. The total cell mass lost from 16 to 72 h after TPA application amounted to about 67% of the newborn cell mass. Thus the hyperplasia occurring after a single TPA treatment results from a considerably enhanced cell proliferation that exceeds concomitant increases in the rates of cell maturation and loss. There is no delayed maturation. The results are also consistent with the chalone theory of epidermal growth regulation, assuming that the G1 chalone is produced during cell maturation.

Expression of gamma-glutamyl transpeptidase activity in the developing mouse tooth, intervertebral disc, and hair follicle.

Expression of gamma-glutamyl transpeptidase (GGT) in the developing mouse tooth, intervertebral disc, and hair follicle was investigated in terms of its localization during ontogenic stages and its association or lack of association with cell proliferation (labeled nuclei after [3H]thymidine injection or metaphase-arrested cells after colchicine injection). The data demonstrate that (a) GGT expression followed a program of activity and localization changes that correlated with the progressive emergence of developmental stages and (b) GGT activity in developing tissues derived either from epithelium (enamel-producing cells and hair follicle cells) or from mesenchyme (intervertebral disc cells) was localized only in mitotically quiescent cellular layers or regions associated with the production of specialized tissue products; however, not all postmitotic regions expressed GGT activity. Although further research is needed to clarify the role of GGT in normal and neoplastic tissues, we conclude that increasing evidence from this and other laboratories implicates GGT as a marker of cell differentiation, cell aging, and/or reduced cell proliferation.

Are datascreen terminals a source of increased PCB-concentrations in the working atmosphere?

The use of polychlorinated biphenyls (PCBs) restricted to closed systems. They are thus still used as dielectric fluids in capacitors and transformers. The hypothesis that skin rashes and excema reported among datascreen operators might be caused by leakage of PCB from components in the screen terminals was investigated. No information about the chemical content of the electric components in the actual datascreen terminals were available. The PCB-concentration in the atmosphere in a workplace where such skin reactions had previously occurred was therefore determined. Air was sampled through 2 or 3 days, during work hours only. A glass fiber backed by an amberlite XAD-2 column was used to trap the sample. The total level of PCBs in this sample was analysed by glass-capillary gas chromatography with electron capture detection. Commercial PCB mixtures were used as standards. PCB-concentrations in the working atmosphere (56-81 ng/M3) were about 50-80 times the level of PCB in samples collected outside the building. Indoor and outdoor samples differed also qualitatively. The indoor samples contained only Aroclor 1242, while the outdoor samples contained a mixture of Aroclor 1242 and 1254. The amounts of PCBs measured were below the safety level for working atmosphere, as recommended by NIOSH in 1977. However, knowing that the samples were collected in an office where there was no known use of PCBs, the levels found were unexpectedly high. Further investigations should therefore be undertaken to clarify whether datascreen terminals might be sources of PCB-contamination.

Ornithine decarboxylase activity in chemically induced mouse skin papillomas.

The activity levels of L-ornithine carboxy-lyase (ODC) (E.C. 4.1.1.17) and S-adenosyl-L-methionine carboxy-lyase (SAM-D) (E.C.4.1.1.50) were determined in individual papillomas induced in mouse skin by a two-stage technique, and in normal mouse epidermis. Cycloheximide treatment abolished both enzyme activities. In normal epidermis the ODC activity was barely detectable, whereas the tumors exhibited high levels of ODC. Levels of SAM-D activity above those of normal epidermis were detected in some papillomas, but in contrast to ODC the SAM-D activity levels were not consistently increased in skin tumors. By pooling a great number of papillomas, the variations in ODC and SAM-D activities between different papillomas could be minimized so that reliable measurements of the biological half-lives of ODC and SAM-D in the tumors were obtained using cycloheximide treatment. The half-life of SAM-D in squamous papillomas was 45 min, almost identical to the 41 min half-life of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced level of this enzyme in normal mouse epidermis. In contrast, the ODC activity of the mouse skin papillomas declined at a rate similar to that in TPA-treated epidermis for only the first 15-20 min after cycloheximide injection. Thereafter, at time points when protein synthesis was approximately 90% inhibited, the ODC activity reverted to high levels. These results show that the high level of ODC activity in squamous papillomas is stabilized. This observation is compatible with the hypothesis that the control mechanism of the ODC activity level in these tumors is severely deranged. This change in polyamine turnover pattern may be related to altered differentiation of the epidermal cells, which constitute the main bulk of cells in these tumors.

Effect of retinoic acid pretreatment on 12-O-tetradecanoylphorbol-13-acetate-induced cell population kinetics and polyamine biosynthesis in hairless mouse epidermis.

A single topical application of 17 nmol 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to the skin of hairless mice induces characteristic transient alterations in the epidermal cells turnover and maturation (0.96 h), associated in time with characteristic changes in the activities of L-ornithine carboxy-lyase (E.C. 4.1.1.17) (ODC) and S-adenosyl-L-methionine carboxy-lyase (E.C.4.1.1.50) (SAM-D) and in the accumulation of polyamines. The effects on these responses of local pretreatment of the skin with retinoic acid 1 h prior to TPA were investigated at selected time points. Retinoic acid inhibited the TPA-induced ODC activity and the ensuing accumulation of putrescine, but did not alter the TPA-induced SAM-D activity or the molar ratio of spermidine/spermine. This pretreatment also decreased in number of dividing basal cells in the first TPA-induced synchronized wave of proliferating cells. However, during the subsequent period of proliferation, the number of dividing cells in the retinoic acid pretreated group was comparatively increased. Hence, at four levels of retinoic acid (0.17, 1.70, 17.0 and 170 nmol), which all inhibited the TPA-induced ODC effectively, there was no change in the total number of basal cells that divided during 16-48 h after TPA-application. Theory is put forward the retinoic acid might exert its antitumorigenic effect during tumor promotion with TPA by interfering with the rate and/or quality of epidermal cell maturation, rather than by inhibiting cell proliferation.

Mouse skin: a useful model system for studying the mechanism of chemical carcinogenesis.

Mouse skin has a long history as a useful model for the study of the mechanism of carcinogenesis (6). In particular, the availability of specific diterpene esters has made possible rapid progress in understanding the mechanism of tumor formation (4,6,8,19,36,41), although certain details may be unique to promotion by phorbol esters. Evidence is compatible with an essential role for elevated levels of polyamines in tumor promotion, but other components of phorbol ester action on mouse skin are also essential (27,40,54). These may include the production of dark cells (22), inhibition of maturation (2,19,41), and the elimination of metabolic cooperation (12,57). Factors modifying biochemical processes that are essential to tumor formation produce a parallel effect on tumor formation. Some of these inhibitors act synergistically to inhibit tumor formation (50,55), and knowledge of their action may lead to practical application for the prevention of human cancer.

Inhibition by epidermal extracts of the 12-O-tetradecanoylphorbol-13-acetate induced peak of ornithine decarboxylase activity in the mouse epidermis.

The time course of induction of epidermal ornithine decarboxylase (E.C. 4.1.117) (ODC) activity following a single topical application of 17 nmoles of 12-O-tetradecanoylphorbol-13-acetate (TPA) on hairless mouse skin was established. Prior intraperitoneal (i.p.) administration of a crude epidermal extract prepared from hairless mouse epidermis led to a time-dependent, 50% inhibition of the peak level of TAP-induced ODC activity. Maximum inhibition was observed when the extract was injected 1.5 h before TPA treatment. The crude epidermal extract did not affect ODC activity in vitro. Following the administration of epidermal extracts, the inhibition of the TPA-induced ODC-response correlated positively with the presence of epidermal G2-chalone activity (determined by a stathmokinetic method) whereas myocardial, skeletal muscle, or heat-inactivated epidermal extracts with no epidermal G2-chalone activity, had no effect on TPA-induced ODC activity. These results indicate a possible relationship between ODC-activity and the control of mitotic rate by G2-chalone.

Cell population kinetics in hairless mouse epidermis following a single topical application of 12-O-tetradecanoylphorbol-13-acetate I.

The alterations in the cell population kinetics of mouse epidermis (hr/hr. Oslo strain) following a single topical application of 17 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) were studied using serveral methods simultaneously. During a period from 2-96 h following treatment, 452 mice were used to measure incorporation of tritiated thymidine into DNA (labeling index and specific activity), mitotic index, Colcemid arrested metaphase rates, and flow cytometric DNA measurements. The time course of the kinetic response to TPA could be divided into two different periods. Period I (0-12 h) was characterized by a temporary block of cells in S-phase and mitosis. In period II (12-96 h), partly synchronized cells displayed multiple waves of DNA synthesis and cell division, with a considerable reduction of the cell cycle time from 54 h down to 10-12 h. The resulting hyperplastic epidermis is therefore composed of a relatively high proportion of young, immature cells. Hence, some of the early biochemical alterations in mouse epidermis assumed to be specific for TPA as a tumor promotor, may merely be the result of a rapid population shift.

Changes in epidermal polyamine biosynthesis and specific activity of DNA following a single application of 12-O-tetradecanoyl-phorbol-13-acetate to hairless mouse skin.

A single application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to hairless mouse skin induces increased activity of epidermal L-ornithine carboxy-lyase (E.C.4.1.1.17) (ODC) with a peak at 5 h, and S-adenosyl-L-methionine carboxy-lyase (E.C.4.1.1.50) (SAM-D) with a broad peak at 20-36 h. The temporal sequence of the accumulation of polyamines; i.e. putrescine, spermidine and spermine, and the rate of DNA synthesis was investigated. All four parameters were measured in the same tissue-samples and multiple peaks of DNA synthesis and of individual polyamines were demonstrated. In the period from 0-12 h, there was an initial decrease in the rate of DNA synthesis. In this period changes in the molar ratio of spermidine/spermine were negatively correlated to the rate of DNA synthesis. From 12-48 h, however, changes in the molar ratio of spermidine/spermine had an almost identical time course with rates of change of DNA synthesis. Based on corresponding cell kinetic results it is suggested that the spermidine/spermine ratio reaches a maximum peak during the S-phase of the cell cycle. The relation between the rate of DNA-synthesis and the spermidine/spermine ratio as well as the ordered time sequence for the accumulation of putrescine and the induction of ODC and SAM-D activities, suggest a strong interdependence and a strict regulation of these events in hairless mouse epidermis induced to proliferate by TPA.

Dissociation of increases in levels of 3':5'-cyclic AMP and 3':5'-cyclic GMP from induction of ornithine decarboxylase by the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate in mouse epidermis in vivo.

A single application of 17 nmol of 12-O-tetradecanoyl phorbol-13-acetate (TPA) to mouse skin caused a marked (200- to 400-fold) induction of ornithine decarboxylase (EC 4.1.1.17, L-ornithine carboxy-lyase) activity in mouse epidermal and epidermal-dermal preparations. No change in the basal level of 3':5'-cyclic AMP occurred in epidermal-dermal preparations within 30 min of TPA application. Intraperitoneal injection of the beta-agonist isoproterenol resulted in a dose-dependent accumulation of 3':5'-cyclic AMP occurred in epidermal-dermal preparations within 30 min of TPA application. Intraperitoneal injection of the beta-agonist isoproterenol resulted in a dose-dependent accumulation of 3':5'-cyclic AMP 10 min after injection, but caused no induction of ornithine decarboxylase. When isoproterenol was injected 10 min prior to an application of either 1.7 or 17 nmol of TPA, the magnitude of the ornithine decarboxylase induction was the same as induction with TPA alone. Topical application of 17 nmol of TPA caused no increase in the level of 3':5'-cyclic GMP present in the mouse epidermal-dermal preparations 2-20 min after application. Intraperitoneal injection of 1.75 mumol of dibutyryl 3':5'-cyclic GMP and/or butyryl derivatives of cyclic GMP caused a 6-fold increase in the level of cyclic GMP and/or butyryl derivatives of cyclic GMP in epidermal-dermal preparations within 5 min of injection, and the level remained elevated for at least 20-30 min. This dose of dibutyryl 3':5'-cyclic GMP was incapable of inducing ornithine decarboxylase. Injection of dibutyryl 3':5'-cyclic GMP 5 min before application of 1.7 nmol of TPA or 30 min before application of 17 nmol of TPA did not alter the magnitude of the ornithine decarboxylase induction produced by TPA alone. These results suggest that early increases in the total intracellular levels of either 3':5'-cyclic AMP or 3':5'-cyclic GMP are not part of the mechanism by which TPA induces ornithine decarboxylase in the epidermis.